Ricerc@Sapienza

Permanent neonatal diabetes mellitus (PNDM) is a type of diabetes characterized by the onset of hyperglycemia within the first six months of life. PNDM could be caused by mutations in INS1 gene both recessive and dominant. The dominant mutations affect proinsulin folding and trafficking; misfolded (pro)insulin is retained inside ER and triggers the unfolded protein response (UPR), an adaptive cellular mechanism aimed at restoring protein-folding homeostasis in specialized secretory cells and apoptosis which leads in turn to the swift decline of insulin secretion. Still, how sustained ER-stress can induce pancreatic beta cell apoptosis is not fully elucidated. INS1L39Y40delinsH mutation is one of the dominant mutations associated to PNDM. Actually INS/PNDM patients depend on insulin for survival, which calls for investment into the design of innovative therapeutic strategies.
A proposed new therapeutic approach was based on lentiviral mediated expression of shRNAs designed to allow specific reduction of mutated INS1 expression. For INS1L39Y40delinsH mutation a shRNA capable of reducing mutated expression up to 70% leaving almost unaltered INS1 expression was already selected. Preliminary results indicate that this shRNA could be efficient in promoting the rescue of CHOP expression induced by mutated INS1 to promote apoptosis triggered by ER stress.
The aim of this project is to complete the characterization of the rescue capability of the selected shRNA expressing lentivector analyzing other PNDM-associated intracellular biomarkers, including: XBP1 splicing products and apoptosis markers (by annexin V and PI). Moreover I will assay corrected or not, INS1 mutated cells by UPR-specific PCR microarrays comparing them with wild type cells. If successful, this approach can be applied to obtain mutant allele-specific silencing in IPSC deriving from INS/PNDM patients that could be differentiated into pancreatic beta-like cells in order to rescue the diabetic phenotype.